Cytochrome c oxidase is the terminal enzyme complex responsible for metabolic respiration. It is an oligomeric enzyme which is associated with the inner mitochondrial membrane and functions as an electron carrier from reduced cytochrome c to molecular oxygen. The energy derived from this electron flow is coupled to the production of ATP within the mitochondria. Because of its key role in cellular energy producton, we have undertaken to study the three-dimensional organization of yeast oxidase. Results obtained from this study will help us to understand how this complex assembled and how it functions as an electron transpoprt carrier. The specific aims of the proposal are to: (1) determine the vectoral distribution of oxidase polypeptides across the mitochondrial inner membrane using lactoperoxidase catalyzed iodination, newly described hydrophilic fluorescent azides and monoclonal antibodies; (2) to use monoclonal antibodies in combination with chemical cross-linking and fluorescence energy transfer measurements to determine subunit-subunit interactions and subunit-heme a relationships; (3) to utilize newly developed cross-linking techniques in combination with surface labeling technology to determine the cytochrome c binding domain on cytochrome c oxidase; (4) to use fluorescence and electron spin resonance techniques to establish the orientation of the bound cytochrome c with respect to the heme a and specific subunits of the oxidase; (5) to utilize the recently described technique of fluorescence energy transfer in the rapid diffusion limit to determine heme localizations in the oxidase; the (6) to apply the new technology obtained from the proposal to determine spacial rearrangements which may have occurred in defined functional mutants of yeast cytochrome c oxidase.